Trivisonno et al. Stem Cell Research & Therapy (2020) 11:451 https://doi.org/10.1186/s13287-020-01968-1

REVIEW Open Access Regenerative medicine approaches for the management of respiratory tract fistulas Angelo Trivisonno1†, Dania Nachira2†, Ivo Boškoski3†, Venanzio Porziella2†, Giuliana Di Rocco4, Silvia Baldari4 and Gabriele Toietta4*

Abstract Respiratory tract fistulas (or fistulae) are abnormal communications between the respiratory system and the digestive tract or the adjacent organs. The origin can be congenital or, more frequently, iatrogenic and the clinical presentation is heterogeneous. Respiratory tract fistulas can lead to severely reduced health-related quality of life and short survival. Therapy mainly relies on endoscopic surgical interventions but patients often require prolonged hospitalization and may develop complications. Therefore, more conservative regenerative medicine approaches, mainly based on lipotransfer, have also been investigated. Adipose tissue can be delivered either as unprocessed tissue, or after enzymatic treatment to derive the cellular stromal vascular fraction. In the current narrative review, we provide an overview of the main tissue/cell-based clinical studies for the management of various types of respiratory tract fistulas or injuries. Clinical experience is limited, as most of the studies were performed on a small number of patients. Albeit a conclusive proof of efficacy cannot be drawn, the reviewed studies suggest that grafting of adipose tissue-derived material may represent a minimally invasive and conservative treatment option, alternative to more aggressive surgical procedures. Knowledge on safety and tolerability acquired in prior studies can lead to the design of future, larger trials that may exploit innovative procedures for tissue processing to further improve the clinical outcome. Keywords: Adipose tissue, Fistula, Regenerative medicine, Respiratory tract, Lipotransfer, Mesenchymal stromal cells, Head and neck, Tracheoesophageal fistula, Minimally invasive treatments, Airway defects restoration

Introduction periorbital region to correct a depressed scar [2]. In Fat grafting, referred also as lipotransfer, involves har- 1987, Sydney R. Coleman developed an innovative tech- vesting of adipose tissue, processing of the collected fat nique of liposuction allowing for adipose tissue harvest to eliminate oil, liposuction fluids, and blood compo- under local anesthesia with less extensive damage [3]. nents, and then re-injection of the manipulated tissue The procedure of fat grafting has been broadly explored into the area that needs treatment [1]. The first docu- to repair soft tissue volume loss (reconstructive surgery) mented surgical fat grafting procedure dates back to and to enhance cosmetic appearance (cosmetic surgery) 1893 when Gustav Neuber described the transfer of adi- [3, 4]. More recently, fat grafting has also been used to pose tissue harvested from the forearm into the promote tissue or organ healing (regenerative medicine) [1, 5–7]. Several parameters such as fat preparation, im- * Correspondence: [email protected] plantation techniques, and recipient site may affect graft † Angelo Trivisonno, Dania Nachira, Ivo Boskoski and Venanzio Porziella retention [8]. As a result, in the absence of a general contributed equally to this work. 4Department of Research, Advanced Diagnostic, and Technological consensus on a standardized procedure, the clinical out- Innovation, Translational Research Area, IRCCS Regina Elena National Cancer come of lipotransfer is not always predictable [9, 10]. Institute, via E. Chianesi 53, 00144 Rome, Italy Full list of author information is available at the end of the article

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In 2001, Zuk et al. demonstrated the presence within still require complete elucidation [22]. MSC are believed the adipose tissue of multipotent cells able to differenti- to exert their pro-healing function mainly through the ate in vitro into adipogenic, chondrogenic, myogenic, release of paracrine factors and extracellular vesicles that and osteogenic cells [11]. This discovery provided fur- may stimulate the migration and activation of local ther support for the perspective use of adipose tissue- tissue-specific stem cells that contribute to tissue regen- derived material for regenerative purposes [12, 13]. The eration, promotion of neo-angiogenesis, modulation of isolation of multipotent cells from the adipose tissue inflammatory and immunomodulatory responses, and complex involves several steps: (1) fat digestion by a so- increase of anti-oxidative and anti-apoptotic effects [23, lution containing collagenase, (2) elimination of tissue 24]. Several regenerative medicine clinical trials using debris by filtration, (3) centrifugation to collect the cellu- MSC cell transplant procedures have been performed lar component of the so-called stromal vascular fraction [25]; in particular, treatment of perianal fistulising Crohn’s (SVF), (4) expansion of the isolated cells in culture to disease, a chronic inflammatory disorder of the gastro- obtain adipose tissue-derived mesenchymal cells, and (5) intestinal tract, using cell therapy has been extensively in- flow cytometry analysis for phenotypic characterization vestigated in virtue of the immunomodulatory properties of the isolated cells [14]. According to the definition re- of MSC [26–28]. A phase III study verified the safety and leased by the International Federation for Adipose the efficacy in long-term closure of perianal fistulas by Therapeutics and Science (IFATS) and the International local injection of adipose tissue-derived MSC [29]. Society for Cellular Therapy (ISCT), uncultured SVF Placement of esophageal stent [30] or bioprosthetic cells are a heterogeneous population that includes stro- materials [31] are currently used for the management of mal cells, endothelial cells, erythrocytes, fibroblasts, lym- different esophago-respiratory fistulas. Unfortunately, phocytes, monocyte/macrophages, and pericytes [15, 16]. this kind of surgical intervention often requires long Mesenchymal stromal/stem cells (MSC), referred also as hospitalization and may be associated with a consider- adipose tissue-derived stromal cells (ASC), are character- able risk of adverse events. Recently, the therapeutic effi- ized by rapid plastic adherence in culture; moreover, cacy of the delivery of cell and tissue-based products for they express the phenotypic markers CD90, CD73, the treatment of fistulas of different etiology has been CD105, and CD44, while they are negative for CD45 and studied. We performed a narrative literature review on CD31 expression; in addition, MSC can differentiate into the management of different kinds of fistulas and osteocytes, adipocytes, and chondrocytes in vitro in the esophageal and airway defects through the administra- presence of appropriate inductive media [15]. During the tion of cellular and tissue-based products, as a conserva- course of the years, adipose tissue-derived multipotent tive alternative procedure to more aggressive surgery. cells [11] were named also as stem cells [17], as mesen- We then focus on the possible future directions, in- chymal stromal cells [15] and, more recently, as medi- cluding the potential use of different methods of adi- cinal signaling cells [18], maintaining the MSC acronym pose tissue manipulation, which may provide an [16]. The evolution of the nomenclature reflects a para- opportunity to improve theclinicaloutcomeofthe digm shift on how MSC are believed to exert their thera- procedure. In order to identify the studies evaluating peutic effect in regenerative medicine procedures. In the effects of autologous fat grafting and/or mesen- facts, the term “multipotent stem cells” was originally chymal stromal cell therapy on airway tissue defects, coined to imply that MSC might differentiate into cells we interrogated PubMed, Web of Science, Scopus, which directly participate into tissue healing (building and Google Scholar electronic databases. Moreover, block activity). Several experimental and clinical evi- we consulted the ClinicalTrials.gov trial registry. We dences subsequently indicated that, despite the develop- conducted literature search by combining Medical ment of different strategies aiming at improving cell Subject Headings terms such as “respiratory tract fis- engraftment [19], the number of cells which are actually tula", “mesenchymal stromal cell”, “adipose tissue- able to survive and persist upon transplant, to differenti- derived stromal cells”, “stromal vascular fraction”, ate in vivo and to take part in tissue regeneration are far “lipoaspirate”,and“adipose tissue”.Studieswerenot too low to justify the clinical benefit observed in cell constrained by publication date or publication status. therapy procedures [20]. Therefore, the attention was Only clinical studies written in English were exam- pointed to the ability of MSC, as “medicinally signaling ined. Identified articles were mainly case reports since cells”, to produce trophic, immunomodulatory factors, no clinical study with a large sample size has been either directly or via extracellular vesicles, which might evaluated so far (Table 1). Moreover, the nature of promote tissue regeneration and/or tissue stem cells the disease, as well as the method of processing and homing (paracrine activity) [21]. The exact molecular local administration of the material, predominantly mechanism(s) underlying the regenerative potential as- derived from autologous adipose tissue, varied among sociated with adipose tissue- and cell-based therapies the studies. Trivisonno et al. Stem Cell Research & Therapy (2020) 11:451 Page 3 of 13

Table 1 Reports of therapeutic procedures involving fat or mesenchymal stromal cells for the management of respiratory tract fistulas Condition Intervention Patients enrolled Reference Oroantral fistula Autologous buccal fat pad 1+ 25 [32–34] Pharyngocutaneous fistula Autologous fat 1 + 1 [35, 36] Tracheoesophageal fistula Autologous fat 1 [37] Tracheomediastinal fistula Autologous adipose tissue SVF in fibrin glue 1 [38] Bronchopleural fistula Autologous adipose tissue-derived MSC-seeded matrix graft 1 [39] Autologous bone marrow-derived MSC 1 + 2 [40, 41] Umbilical cord MSC 1 [42] Autologous fat 8 [43] SVF stromal vascular cells (uncultured), MSC mesenchymal stromal cells

Clinical application of adipose tissue-derived Pharyngocutaneous fistula material for the treatment of respiratory tract Pharyngocutaneous fistula (PCF) is a pathological com- fistulas munication involving the digestive tract and the skin of According to the Medical Subject Headings (MeSH) def- the neck. PCF is a quite common complication after inition, a respiratory tract fistula is “an abnormal passage head and neck surgery [49]. Presence of PCF may pro- communicating between any component of the respira- long recovery and delay adjuvant oncologic treatments. tory tract or between any part of the respiratory system The majority of cases are treated with conservative man- and surrounding organs”. If left untreated, respiratory agement in order to promote spontaneous healing, but tract fistulas are associated with high mortality rates approximately 30% of patients require a more aggressive [44–46]. The most common interventional therapy relies surgical intervention. Two different case reports have on stent placement. In addition, more conservative strat- described successful PCF healing by fat grafting in pa- egies based on regenerative medicine approaches have tients undergone to partial pharyngectomy [35, 36]. In also been considered. In the following sections, we particular, in the case report described by Hespe et al., briefly review some of the tissue/cell-based clinical stud- two rounds of autologous fat grafting delivered into the ies described for the management of various types of re- area immediately surrounding the PCF using both blunt spiratory tract fistulas (Table 1). cannulas and 18 gauge needles were performed to achieve complete fistula healing [36]. Conversely, Sapundzhiev et al. reported a case report of a patient ad- Oroantral fistula ministered with autologous fat around the internal open- An oroantral fistula (OAF) is a pathologic communica- ing of the PCF with a Peretti angular injection cannula tion between the oral and the antral cavities. The re- using an endoscopic access to the neopharynx [35]. moval of the maxillary posterior teeth is considered the major cause of OAF development. Small-size OAF tend Tracheoesophageal fistula to heal spontaneously, while surgical intervention is rec- Tracheoesophageal fistulas (TEF) are connections be- ommended for fistulas larger than 3 mm. Taking in con- tween the airway and upper gastrointestinal tract; they sideration of the size of the OAF and the condition of need prompt identification and treatment to prevent re- the surrounding tissues, different therapeutic approaches current and intractable infections due to tracheobron- have been evaluated [47]. Larger defects, such as the chial contamination. TEF are broadly categorized into ones subsequent to tumor resection, may require the use congenital and acquired fistulas, the latter group being of autogenous bone and soft tissue grafts, the placement further divided into nonmalignant and malignant. Con- of allogenous materials or xenografts. Flaps utilizing genital TEF occur in 1 in 3000–5000 live births [50], and local tissue, such as buccal and palatal flaps, can be used they are usually diagnosed within the first year of life, to close moderate-sized defects. In particular, application while presentation in adults is rare [51]. Acquired non- of the buccal fat, a lobulated form of adipose tissue, has malignant TEF are mainly associated with traumatic in- been quite extensively utilized since its description in jury, foreign body or caustic ingestion [52]. The majority 1977 [32–34]. The adipose tissue used to repair OAF is of acquired nonmalignant TEF are mostly due to com- generally coated by the surrounding mucosa in 4 to pression from an inflated endotracheal or tracheostomy 6 weeks, thus promoting complete epithelialization of tube cuff which may occur in approximately 0.5% of pa- the treated area [48]. tients undergoing tracheostomy or intermittent positive Trivisonno et al. Stem Cell Research & Therapy (2020) 11:451 Page 4 of 13

Fig. 1 Iatrogenic tracheoesophageal fistula after emergency orotracheal intubation. Computed tomography (CT) images showing a large tracheoesophageal fistula (red arrow) in axial (a) and sagittal plane (b). Endoscopic image (c) of the fistula (red dashed line) between esophageal lumen (blue arrow), with a nasogastric tube inside (blue star), and tracheal lumen (red arrow) pressure ventilation [53] (Fig. 1). Occasionally, nonma- therapies may attenuate chemotherapy-induced tissue lignant acquired TEF may arise from local inflammation injuries [56]. and infection, such as tuberculosis and granulomatous Small size TEF may close spontaneously, while fistulas infection [54]. over 20 mm in size are associated with poor survival Malignant acquired TEF have been associated with [57]. Therefore, prompt therapeutic intervention is several types of cancers. In particular, TEF incidence has needed in order to arrest the contamination of the air- been reported as 4.5% following primary malignant way and enabling normal oral alimentation. Different ap- esophageal tumors and 0.3% in primary malignant lung proaches have been developed for the management of tumors [55]. Tumor invasion and cancer-related tissue both acquired non-malignant [57] and malignant TEF necrosis may contribute to the pathogenesis of malig- [44, 46, 55]. Surgical interventions include esophageal nant TEF. In addition, also chemoradiotherapy and stent placement, bypass, resection, and surgical repair. anti-angiogenic therapy, affecting local architectural Conservative treatments, alternative to surgical proce- and vascular tissue changes, can increase the risk of dures, mainly consist of supportive care to prevent con- TEF formation [46](Fig.2). In this regard, cell-based tamination of the respiratory tract. Moreover, use of

Fig. 2 Neoplastic bronchoesophageal fistula after inductive radiotherapy. a CT image of the fistula between esophageal lumen and left main bronchus (red arrow). b Bronchoscopic view of the same fistula (blue arrow) on the membranous side of left main bronchus Trivisonno et al. Stem Cell Research & Therapy (2020) 11:451 Page 5 of 13

autologous tissue-assisted regenerative procedure may promote healing [39]. The treated patient remained represent a valuable therapeutic option. In this regard, it asymptomatic during the clinical follow-up of 1.5 years. has been described a case report of a 55-year old man af- Díaz-Agero Álvarez et al. described the treatment of two fected by congenital TEF successfully treated with local patients suffering from BPF by bronchoscopic adminis- injection of autologous fat using a pressurized injection tration of adipose tissue-derived stromal cells (ASC) iso- device [37]. Long-term complete healing was observed lated by collagenase digestion and not expanded in after two sessions of administration of autologous fat culture [41]. One patient, affected with a 6-mm diameter and the patient remained asymptomatic more than 10 BPF, was administered with 4.0 × 106 ASC leading to years. 80% closure of the fistula. Six months later, the proced- ure was repeated with the administration of additional Tracheomediastinal fistula 5.0 × 106 ASC to achieve full healing. The second A tracheomediastinal fistula (TMF) is a communication patient, who had a 3-mm diameter fistula, received 1.3 × between the trachea and the mediastinum. TMF forma- 107 ASC in a single procedure. Patients were observed tion is rare and generally associated with airway tumors. for a 3-year follow-up, and no treatment-related adverse Díaz-Agero Álvarez described a case report of TMF, effects were reported [41]. Recently, Zeng et al. subsequent to endoscopic laser therapy of tracheal can- described a case report of successful closure of a BPF cer, treated with bronchoscopic administration of au- (5 × 2 mm) resulting from lobectomy, treated by admin- tologous ASC in fibrin glue suspension [38]. In istration through a flexible bronchoscope of 2.0 × 107 particular, autologous ASC were isolated by collagenase umbilical cord MSC around the fistula [42]. A computed digestion from 150 ml of lipoaspirate. Then, approxi- tomography scan performed 6 months after the treat- mately 5.0 × 106 cells were mixed in fibrin glue and ment revealed fistula healing and the BPF did not re- injected through a bronchofibroscope into the cavity of lapsed during the 2-year follow-up. A different approach a 2-cm2 TMF. One-year follow-up showed complete has been evaluated by Huramoto et al. in lung cancer closure of the fistula with re-epithelialization and neo- patients undergoing lobectomy. The authors suggest that vascularization of the area (Fig. 3)[38]. the use of isolated pericardial fat tissue to close the bronchial stump might prevent the occurrence of BPF Bronchopleural fistula [60]. Recently, endoscopic administration of autologous A bronchopleural fistula (BPF) is defined as a patho- fat was performed for the treatment of BPF in 8 patients logical communication between the bronchial tree and and resolution was observed in all cases [43]. the pleural space [58]. BPF is a severe postoperative complication of pneumonectomy or other pulmonary re- Clinical application of adipose tissue-derived section interventions with high rates of morbidity and material to promote tissue regeneration in the mortality (Fig. 4). Therefore, surgical or bronchoscopic oropharyngeal tract interventions are needed to promote BPF closure. In the next sections, we present a brief overview of the As conservative alternative to more traumatic surgical studies of reconstructive/regenerative surgery assessing procedures, administration of mesenchymal cells has local administration of fat or adipose tissue-derived mes- been performed in order to promote healing of the tis- enchymal stromal cells to restore tissue loss or damage sue surrounding the fistula [42, 59]. In particular, Pet- in the oropharynx (Table 2). rella et al. described an approach of autologous bronchoscopic perilesional transplantation of ten million Tracheoesophageal puncture bone marrow-derived mesenchymal cells for the treat- Tracheoesophageal puncture (TEP) with voice prosthesis ment of a small-caliber (3 mm) BPF developed in a 42- placement is an extensively used technique to restore year-old man after right extra pleural pneumonectomy vocal function in patients undergoing total laryngectomy for malignant mesothelioma [40]. Aho et al. described a and pharyngolaryngectomy. One of the most frequent case report of a 66-year-old patient with a large (1.5 cm) complications of this procedure, which usually requires BPF treated with a matrix graft seeded with autologous to replace the voice prosthesis, is enlargements of the mesenchymal stem cells. Cells were obtained by collage- puncture, with leakage of saliva or food [76]. Adminis- nase digestion from autologous adipose tissue and tration of autologous fat around the puncture has been underwent three passages of amplification in vitro, and described as an effective and safe procedure, which al- finally, 2.5 × 107 MSC were seeded on a matrix of syn- lows the conservation of the voice prosthesis, by pro- thetic bio-absorbable poly(glycolide:trimethylene carbon- moting the increase of the thickness of the ate) copolymer under Good Manufacturing Practices tracheoesophageal wall [61]. In particular, 4 out of the (GMP) procedures. Five days after cell seeding, the 10 treated patients maintained long-term (up to 65 matrix graft was surgically placed over the BPF to months) tracheoesophageal speech with no leakage. Trivisonno et al. Stem Cell Research & Therapy (2020) 11:451 Page 6 of 13

Fig. 3 Bronchoscopic and CT images from the region of the fistula. a Bronchoscopic image recorded before cell therapy. The fistula can be seen on the anterior tracheal wall which had been totally destroyed after the laser treatment of the tumor. The entrance was about 10 mm in diameter and the bronchoscope could pass through it. Inset: Anthracotic mediastinal lymph nodes as seen through the wall of the fistula. b CT image recorded before cell therapy. The fistula was situated between the trachea and a pretracheal mediastinal cavity with an area of 2 cm2, next to the superior vena cava and pulmonary artery, near the ascending aorta. c Bronchoscopic image recorded 1 year after cell therapy. The entrance to the fistula was much smaller (diameter 3–4 mm). Inset: The walls of the fistula were covered with “new” epithelium and vessels as a result of neovascularisation and epithelialisation. d CT image from the same region of the fistula 1 year after cell therapy. One year after treatment the cavity had disappeared. e CT image from the region of the fistula recorded 1 year after cell therapy. This image is the only one to show remnants of the previous fistulous tract. It is clear that the fistula had closed. *, small depression; VC, superior vena cava; AOa, ascending aorta; AOd, descending aorta; PA, right pulmonary artery; LPA, left pulmonary artery. Reproduced with permission from Díaz-Agero Álvarez et al. [38]

Hypertrophic tracheostomy scar adipose tissue has been employed by Mazzola et al. Hypertrophic scar formation at the site of tracheostomy resulting in valuable cosmetic results and in improved is quite frequent. The scar tissue may attach to the tra- skin quality and texture [62]. All 10 treated patients en- chea causing discomfort during the act of swallowing. rolled in the study achieved satisfying esthetic and func- Several surgical options have been described for hyper- tional improvements. trophic scar ablation [77]. Fat, adipose tissue-derived mesenchymal stromal cells, and stromal cell-derived fac- Head and neck reconstruction after radiotherapy tors possess antifibrotic functions which exert a positive Reconstructive surgery may be required for functional role in difficult scar treatment [78]. A minimally invasive and cosmetic soft tissue restoration in patients with head procedure for the treatment of post-tracheostomy hyper- and neck cancers. Actually, treatment of head and neck trophic scar by means of intralesional administration of cancers may require the surgical removal of a large Trivisonno et al. Stem Cell Research & Therapy (2020) 11:451 Page 7 of 13

Fig. 4 Bronchopleural fistulas after pulmonary lobectomy and pneumonectomy for lung cancer. a Bronchoscopic view of the fistula (and muco- purulent secretions) in the inferior right bronchial stump; b endoscopic view of the bronchial stump fistula after right pneumonectomy amount of tissue surrounding the tumor; moreover, ad- collectively performed for the management of VPI in juvant therapeutic irradiation may result in extensive tis- more than 250 patients [66]. Recently, the procedure sue damage and induction of radiation-induced skin was described for additional 11 adult patients [67]. Based fibrosis. Administration of autologous fat has been eval- on these preliminary studies, the procedure of injection uated as a suitable method to achieve both esthetic and pharyngoplasty with autologous fat could be considered functional reconstruction in head and neck oncologic pa- as a safe and effective treatment option for mild VPI. tients, partially restoring volume loss, reducing excessive scar formation and radiation-induced skin fibrosis in the Vocal fold scars treated areas [63–65, 79]. More than 60 patients have Vocal fold scars are scarring and fibrotic formations on been treated using autologous fat administration in three the layer of the vocal cord. Preclinical studies support different studies [63–65, 79]. Some concerns have been the rationale for using cell therapy for the treatment of raised about the possibility of administered fat to promote vocal fold scarring which may occur as a result of surgi- residual tumor cell invasion and metastasis [80]. Prelimin- cal or iatrogenic injury [82]. A clinical team at the Hopi- ary data obtained using head and neck cancer cell lines taux De Marseille, France, promoted a clinical trial both in vitro and in vivo suggest that the procedure may entitled “Innovative treatment for scarred vocal cords by be safe, but further investigation performed on patient- local injection of autologous stromal vascular fraction” derived tumor samples is needed [81]. (NCT02622464), described the first clinical case report [68], and have recently published the results on add- Velopharyngeal insufficiency itional 8 patients [69]. The therapeutic intervention con- Velopharyngeal insufficiency (VPI) occurs when there is sisted in autologous adipose tissue harvest, enzymatic incomplete velopharyngeal closure. As reviewed by Nigh digestion, isolation of ASC under GMP conditions, and et al. administration of autologous fat has been same-day local administration at the laryngeal level of

Table 2 Reports of therapeutic procedures involving fat or adipose tissue-derived cells to promote tissue regeneration in the oropharyngeal tract Condition Intervention Patients enrolled Reference Tracheoesophageal puncture Autologous fat 10 [61] Hypertrophic tracheostomy scar Autologous fat 10 [62] Radiation-induced fibrosis and volume defects in head and neck oncology Autologous fat 38 + 11 + 12 [63–65] Velopharyngeal insufficiency Autologous fat 11 + 251 [66, 67] Vocal fold scars Autologous SVF 8 + 1 [68, 69] Autologous fat 24 [70] Autologous nanofat and microfat 7 [71] Unilateral laryngeal nerve paralysis Autologous fat > 90 [70, 72–75] SVF adipose tissue-derived stromal vascular fraction cells (uncultured) Trivisonno et al. Stem Cell Research & Therapy (2020) 11:451 Page 8 of 13

2.2–13.6 × 106 viable uncultured cells. Follow-up analysis Future directions performed at 12 months indicated an improvement in We have summarized in Tables 1 and 2 a number of the voice handicap index score without serious adverse published clinical studies on promotion of tissue healing events causally related to the treatment. In addition, in the respiratory tract applying a conservative, regenera- Cantarella et al. described the results of the injection of tive medicine-based approach. Most of the evidence- approximately 0.2 to 3 ml of autologous fat in 24 pa- based data have been collected from single case reports tients with vocal fold scarring [70]. More recently, the or series of case studies. As a matter of facts, the design same authors described the treatment of 7 patients with of larger, multisite clinical trials is hampered by the rela- vocal fold scarring by nanofat and microfat grafting [71]. tively small number of affected individuals that can be In particular, in this group of patients, microfat was ad- enrolled and by the lack of clinical institutions which ministered deeply in the vocal fold and nanofat emulsion have sufficient knowledge and resources for innovation was injected in the most superficial layer of the vocal implementation in this field. Accordingly, the number of fold in the scarred tissue. Follow-up analysis performed clinical trials which have been performed or are cur- at 3 months indicated improvements in the voice handi- rently ongoing is still limited (Table 3). Bone marrow, cap index. adipose tissue, and umbilical cord blood are the most frequently utilized sources of MSC for clinical trials [23], including the ones described for fistula healing and tis- Unilateral recurrent laryngeal nerve paralysis sue regeneration in the oropharynx (Table 3). Bone mar- Unilateral recurrent laryngeal nerve paralysis may occur row and adipose tissue are replenishable sources of MSC secondary to injury of the recurrent laryngeal nerve due suitable for autologous transplant [85]. Bone marrow to cancers, trauma, and surgery. In 1991, Mikaelian et al. collection is an invasive procedure, while subcutaneous published a preliminary report describing a procedure of adipose tissue can be easily harvested [86]. Adipose tis- autologous fat injection into a paralyzed vocal cord in 3 sue contains up to 500 more MSC cells than an equiva- patients affected by unilateral vocal cord paralysis [72]. lent amount of bone marrow; moreover, adipose tissue- Since then the procedure has been performed on several derived MSC can be easily expanded in vitro since they additional cases describing long-term (> 1 year) improve- have higher proliferation rate compared to bone ment of vocal parameters after a single fat injection [70, marrow-derived MSC [87]. In addition, MSC derived 73–75, 83, 84]. In particular, in a clinical trial from adipose tissue promote stronger immunosuppres- (NCT02904824), a group of patients was treated by sive effects than MSC isolated from other sources [88]. administration of adipose tissue and a second group with MSC in umbilical cord are rare but can be amplified the same amount of adipose tissue in presence of a not- in vitro given that can undergo to more cell divisions well quantified amount of ASC (cell-assisted lipotrans- than MSC from adult tissues before reaching senescence. fer) [74]. However, the study was inconclusive in Optimal storage of the cryopreserved umbilical cord tis- determining any definite difference between the clinical sues or MSC is required for autologous use. In general, outcomes of the two groups [74]. easiness of collection and processing make adipose tissue

Table 3 Clinical Trials involving tissue- and cell-based therapy approaches to promote fistula and tissue regeneration in the oropharynx Condition Intervention ClinicalTrials.gov Identifier1 Tracheoesophageal fistula, Adipose-derived stromal vascular fraction for aero-digestive fistulae NCT03792360 bronchoesophageal fistula, tracheal fistula Bronchial fistula Human amniotic epithelial cells for treatment of bronchial fistula NCT02959333 Bronchopleural fistula Umbilical cord mesenchymal stem cells for treatment of bronchopleural fistula NCT02961725 Enterocutaneous fistula Stromal vascular fraction for treatment of enterocutaneous fistula NCT01584713 Dysphonia Innovative treatment for scarred vocal cords by local injection of autologous NCT02622464 stromal vascular fraction Vocal cord paralysis, unilateral Injection laryngoplasty using autologous fat enriched with adipose-derived regen- NCT02904824 erative stem cells Hoarseness, dysphonia, aphonia, vocal fold; A study of local administration of autologous bone marrow mesenchymal stromal NCT04290182 scar cells in dysphonic patients with vocal fold scarring Vocal fold; scar Pilot study of bone marrow stem cell treatment of patients with vocal fold scarring NCT01981330 1Source: https://clinicaltrials.gov/, accessed July 2020 Trivisonno et al. Stem Cell Research & Therapy (2020) 11:451 Page 9 of 13

the best source of material suitable for clinical studies the effective treatment of rectovaginal fistula in Crohn’s aiming at the promotion of tissue healing in the respira- disease by administration of autologous adipose tissue- tory tract. Collectively, the data acquired so far generally derived MSC [92]. Subsequently, additional phase I to confirm the safety and suggest the occasional clinical ef- III clinical trials, collectively enrolling more than 300 ficacy of the delivery of adipose tissue-derived material Crohn’s patients, have been performed indicating that for the treatment of respiratory-digestive tract fistulas cell transplantation is safe and effective [26, 27, 29, 93]. [89]. Nonetheless, there is a strong need to optimize and Isolation of MSC cells mainly relies on collagenase di- standardize the protocols to process adipose tissue in gestion of the fat collected by lipoaspiration [14]; from order to improve the reproducibility of the procedure. the regulatory point of view enzyme-based protocols Moreover, the follow-up conditions must be clearly de- cannot be considered “minimal manipulation” and there- fined to better evaluate the beneficial effects of the fore the manufacturing procedures are subjected to the treatment. regulation applied for the Advanced Therapies Medicinal The majority of the collected clinical information is Products (ATMPs) [94]. Moreover, amplification in based upon studies exploiting administration of unpro- culture of MSC requires GMP conditions, is time- cessed autologous adipose tissue collected by liposuction consuming, and is associated with high cost and (Fig. 5; Tables 1 and 2)[90]. Adipose tissue is mainly regulatory burden. Therefore, alternative, enzyme-free composed of adipocytes, which constitute more than strategies to obtain a ready-to-use adipose tissue-derived 90% of its volume; additional components of the stromal material have been developed [95] and administration of vascular fraction (SVF) include mesenchymal/stromal mechanically isolated adipose tissue SVF has been cells (MSC), preadipocytes, fibroblasts, endothelial cells, performed for the treatment of different pathologies vascular smooth muscle cells, resident monocytes/mac- [96]. In particular, administration of homogenized adi- rophages, and lymphocytes. MSC have a perivascular pose tissue has been recently proved effective in the origin; accordingly, MSC content is higher in vascular- treatment of perianal fistulas in patients with Crohn’s ized hypodermic adipose tissue [86]. In consideration of disease [97–99], providing a suitable alternative to MSC the presence within the adipose tissue of cells able to administration [100]. differentiate and to promote tissue regeneration acting We believe that the strategy of micro-fragmented adi- in a paracrine fashion [22, 91], fat has been recently pose tissue transplantation, considered for the promo- reconsidered not only as a simple physical filler for cos- tion of healing of anal fistulas, may be effectively applied metic surgery procedures, but also as a source of “medi- also for the management of fistulas affecting the upper cinally signaling cells” [18]. Therefore, as an alternative esophageal tract (Fig. 5). Administration of micro- to or in conjunction with lipotransfer, transplantation of fragmented adipose tissue-derived stromal fraction tissue adipose tissue-derived cells has been clinically evaluated (tSVF) has numerous advantages compared to lipotrans- for the treatment of a variety of regenerative purposes fer or administration of MSC: tSVF can be obtained [25]. In particular, in 2003, Garcia-Olmo firstly reported from lipoaspirate by non-enzymatic methods with

Fig. 5 Schematic representation (not in scale) of the therapeutic intervention procedure proposed for the management of respiratory tract fistulas by endoscopic delivery of autologous adipose tissue-derived material. Some templates to create this figure are used/adapted from Servier Medical Art (https://smart.servier.com/), available under a Creative Commons Attribution 3.0 Unported License Trivisonno et al. Stem Cell Research & Therapy (2020) 11:451 Page 10 of 13

minimal manipulation; the procedure is rapid and cost- In addition, accurate 3D-printed patient-personalized effective and can be performed intra-operatively [95]; in stent, based on 3D reconstruction of the fistula image, can tSVF, the relative number of MSC per tissue volume is be created, as assessed for the treatment of enterocuta- higher than in adipose tissue, since adipocytes, red blood neous fistulas [115]. Clinical translation of preclinical re- cells, oil, and aqueous fractions have been discarded; ho- search on tissue engineering for airways defects has been mogenized tSVF can be precisely administered at the site so far limited but the rapid pace of the technological de- of tissue damage through a 25-G needle; compared to velopments in tissue engineering and in 3D bioprinting enzymatically derived SVF, micronized tSVF retains the can anticipate future therapeutic opportunities [116]. native extracellular matrix and perivascular structures, reducing induction of anoikis upon transplant [101]; Conclusions microfat lobules are less prone to oxidative stress com- Respiratory tract fistulas may develop as complications pared to unprocessed fat, thus improving graft retention in various surgical interventions, trauma, and accidental since oxidative damage has a detrimental effect on sur- foreign body and caustic ingestion or, rarely, may be vival of transplanted cell [102] and adipose tissue [103]. congenital. Small size (< 2 mm) fistulas generally heal Several preclinical studies assessed the efficacy of MSC spontaneously, while large caliber fistulas may be associ- therapy for laryngotracheal stenosis [104]. Therapeutic ated with severe, life-threatening complications. We benefit associated to MSC transplant is likely attribut- reviewed several case reports suggesting that endoscopic able to the secretion of soluble factors and to the release local delivery of adipose tissue/MSC may represent a of extracellular vesicles (EVs) [22]. Indeed, administra- moderately invasive and a relatively safe treatment op- tion of conditioned medium collected from MSC cell tion, alternative to aggressive surgery, to promote fistula culture may be instrumental in stimulating resident healing. One possible strategy which may provide a fur- bronchioalveolar stem cells, supporting tissue regener- ther therapeutic advancement could be represented by ation in the respiratory system [105]. Moreover, admin- the delivery of micronized adipose tissue, which can be istration of extracellular vesicles produced by adipose obtained with minimal manipulation [95]. However, tissue MSC mixed in a thermoresponsive gel has been much work remains to be performed before successfully shown to promote esophageal fistula healing in a porcine translation of clinically competitive cell- and tissue- model [106]. Accordingly, the delivery of MSC secre- based new therapies for respiratory tract fistula healing. tome has been proposed as a therapeutic strategy for In particular, standardization of the procedures, lung injury and acute and chronic diseases [107, 108]. optimization of clinical trial design, and guidance in Notably, micro-fragmented fat has improved paracrine follow-up analysis are needed in order to assess the anti-inflammatory, anti-fibrotic, and pro-angiogenic pro- long-term occlusion of the fistulas in the treated prieties instrumental for supporting tissue regeneration patients. compared to cultured MSC [109]. Combination of autologous mesenchymal stromal cells Abbreviations and tissue-engineered scaffolds is an interesting and rap- ASC: Adipose tissue-derived stromal cells; ATMPs: Advanced therapies medicinal products; BPF: Bronchopleural fistula; EV: Extracellular vesicles; idly evolving approach in the regenerative medicine GMP: Good Manufacturing Practices; MSC: Mesenchymal stromal cells; arena, potentially suitable also to support the healing of OAF: Oroantral fistula; PCF: Pharyngocutaneous fistula; SVF: Stromal vascular large-size fistulas and partial or long-segment defects of fraction; TEF: Tracheoesophageal fistula; TEP: Tracheoesophageal puncture; the esophagus [110]. The ideal scaffold should be bio- TMF: Tracheomediastinal fistula; VPI: Velopharyngeal insufficiency compatible and biodegradable, with a degradation rate Acknowledgments similar to the tissue regeneration time. Placement of Not applicable. tissue-engineered graft has been mostly described in pre- clinical models: for instance, the use of suture filament Authors’ contributions — embedded with adipose tissue-derived MSC has been Conceptualization, A.T., D.N., I.B., V.P., and G.T.; writing original draft preparation, G.T. and G.D.R.; writing—review and editing A.T., D.N., S.B., and used to promote fistula healing in a rat model [111]; G.T. All authors have read and agreed to the published version of the promotion of esophageal anastomotic leakage healing manuscript. has been achieved in rabbits by administration of fibrin scaffolds including MSC [112]; synthetic polyurethane Funding This research was funded in part by the Italian Ministry of Health (Ricerca electro-spun grafts seeded with autologous MSC have Corrente – IRCCS IRE) and Istituto Regina Elena Cinque per Mille (5×1000 been tested for esophageal tissue remodeling in pigs 2015 to Gabriele Toietta). The funding sources had no role in study design; [113]. To circumvent possible biocompatibility prob- in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. lems, esophagus-like scaffold-free structures embedded with MSC suitable for esophageal repair have been gen- Availability of data and materials erated by 3D bioprinting and transplanted in rats [114]. Not applicable. Trivisonno et al. Stem Cell Research & Therapy (2020) 11:451 Page 11 of 13

Ethics approval and consent to participate 16. Viswanathan S, Shi Y, Galipeau J, Krampera M, Leblanc K, Martin I, et al. Not applicable. Mesenchymal stem versus stromal cells: International Society for Cell & Gene Therapy (ISCT®) Mesenchymal Stromal Cell committee position Consent for publication statement on nomenclature. Cytotherapy. 2019;21(10):1019–24. Not applicable. 17. Gimble J, Guilak F. Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy. 2003;5(5):362–9. Competing interests 18. Caplan AI. Mesenchymal stem cells: time to change the name! Stem Cells – The authors declare that they have no competing of interests. Transl Med. 2017;6(6):1445 51. 19. Baldari S, Di Rocco G, Piccoli M, Pozzobon M, Muraca M, Toietta G. Author details Challenges and strategies for improving the regenerative effects of 1Department of Surgical Science, University of Rome “La Sapienza”, Viale mesenchymal stromal cell-based therapies. Int J Mol Sci. 2017;18(10):2087. Regina Elena 324, 00161 Rome, Italy. 2Department of General Thoracic 20. von Bahr L, Batsis I, Moll G, Hägg M, Szakos A, Sundberg B, et al. Analysis of Surgery, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Largo tissues following mesenchymal stromal cell therapy in humans indicates A. Gemelli 8, 00168 Rome, Italy. 3Digestive Endoscopy Unit, Fondazione limited long-term engraftment and no ectopic tissue formation. Stem Cells. – Policlinico Universitario Agostino Gemelli IRCCS, 00168 Rome, Italy. 2012;30(7):1575 8. 4Department of Research, Advanced Diagnostic, and Technological 21. Rendra E, Scaccia E, Bieback K. Recent advances in understanding Innovation, Translational Research Area, IRCCS Regina Elena National Cancer mesenchymal stromal cells. F1000Res. 2020;9:Faculty Rev-156. Institute, via E. Chianesi 53, 00144 Rome, Italy. 22. Jimenez-Puerta GJ, Marchal JA, López-Ruiz E, Gálvez-Martín P. 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